RESUMO
Objective:To study the structure of the human THOC1-THOC2-THOC3 subcomplex by negative-staining electron microscopy. Methods:The recombinant protein components were co-expressed in cultured insect cells, and the protein complex was isolated via sequential Ni-NTA and Strep-Tactin affinity purification followed by Superose gel filtration chromatography. The purified protein sample was then subjected to negative-staining electron microscopy and single particle image analysis. Results:The recombinant human THOC1-THOC2-THOC3 complex with high purity and good homogeneity was obtained by using a tandem affinity purification scheme with Ni-NTA and streptavidin-based chromatography. Preliminary study on the structure of human THOC1-THOC2-THOC3 subcomplex was achieved by electron microscopy using negative staining with uranium formate. Conclusion:A low resolution model of human THOC1-THOC2-THOC3 subcomplex was achieved by single particle reconstruction.